Compositions And Methods For Treating Ischemia And Ischemia-Reperfusion Injury

ABSTRACT

The present invention relates to compositions comprising a substantially pure compound represented by Structural Formula I: 
     
       
         
         
             
             
         
       
     
     and methods of using such compounds to activate cytoprotective kinases. The values and preferred values of the variables in Structural Formula I are defined herein.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/283,844 filed May 21, 2014, which is a continuation of U.S.application Ser. No. 13/041,001, filed Mar. 4, 2011, now U.S. Pat. No.8,772,249, issued Jul. 8, 2014, which is a continuation of U.S.application Ser. No. 12/466,170, filed May 14, 2009, now U.S. Pat. No.7,928,067, issued Apr. 19, 2011.

The entire teachings of the above applications are incorporated hereinby reference.

BACKGROUND OF THE INVENTION

In the United States, cardiovascular disease is the leading cause ofdeath for both men and women. More than one million people suffer fromheart attacks every year in the United States alone. Cardiac ischemia, acondition characterized by reduced blood flow and oxygen to the heartmuscle, or myocardium, is one hallmark of cardiovascular disease thatcan ultimately lead to a heart attack, or myocardial infarction.Cardiovascular disease can also result in restricted blood flow andreduced oxygen supply to other areas of the body resulting in ischemicinjuries to various organs and tissues, including the brain, which canlead to stroke.

Re-establishment of blood flow, or reperfusion, and re-oxygenation ofthe affected area following an ischemic episode is critical to limitirreversible damage. However, reperfusion also brings potentiallydamaging consequences, such as reperfusion injury, which is caused bythe restoration of coronary blood flow after an ischemic episode andresults from the generation and accumulation of reactive oxygen andnitrogen species during reperfusion. Ischemia-reperfusion injury isbiochemically characterized by a depletion of oxygen during an ischemicevent, a resultant increase in intracellular calcium levels, followed byreoxygenation and the concomitant generation of reactive oxygen speciesduring reperfusion (Piper, H. M., Abdallah, C., Schafer, C., The firstminutes of reperfusion: a window of opportunity for cardioprotection.Annals of Thoracic Surgery 2003, 75:644; Yellon, D. M., Hausenloy, D.J., Myocardial reperfusion injury. New England Journal of Medicine 2007,357:1121). Reperfusion injury may be responsible for as much as 50% ofthe damage to the heart following a myocardial infarction (Yellon, D.M., Hausenloy, D. J., Myocardial reperfusion injury. New England Journalof Medicine 2007, 357:1121).

The prevalence of cardiovascular disease in the United States, andthroughout the world, necessitates the development of therapies andtherapeutic agents that can effectively prevent, reduce, or counteractischemia and ischemia-reperfusion injury resulting from a heart attackor stroke. Current therapies for treating ischemia andischemia-reperfusion injury caused by myocardial infarction, such asmechanical ischemic preconditioning, have proven to be clinicallyimpractical, while other therapies, such as antagonists to block theinflux of calcium and scavengers of reactive oxygen species, haveyielded disappointing clinical outcomes (Otani, H., Ischemicpreconditioning: From molecule mechanisms to therapeutic opportunities.Antioxidants & Redox Signaling, 2008, 10:207; Yellon, D. M., Hausenloy,D. J., Myocardial reperfusion injury. New England Journal of Medicine2007, 357:1121).

Thus, there is a significant need for new and more effective therapiesand therapeutic agents for the treatment of ischemia andischemia-reperfusion injuries resulting from cardiovascular disease andother conditions.

SUMMARY OF THE INVENTION

The invention described herein addresses a need for treating ischemiaand ischemia-reperfusion injury, including myocardial ischemia andischemia-reperfusion injury, by activating kinases involved in cellsignaling pathways that inhibit apoptosis and by scavenging reactiveoxygen species. In particular, the present invention relates tocompositions comprising the disclosed compounds, or pharmaceuticallyacceptable salts thereof, and their effective use as activators ofcytoprotective kinases.

In one embodiment, the invention relates to compositions comprising asubstantially pure compound represented by Structural Formula I:

wherein R1 and R2 are each independently H or a hydrolyzable group, or apharmaceutically acceptable salt thereof.

In another embodiment, the invention relates to compositions comprisinga substantially pure compound represented by Structural Formula II:

or a pharmaceutically acceptable salt thereof.

In a further embodiment, the invention relates to compositionscomprising a substantially pure compound represented by StructuralFormula III:

In another embodiment, the invention relates to a compositioncomprising: i) a pharmaceutically acceptable carrier or diluent; and ii)a substantially pure compound represented by Structural Formula I:

wherein R1 and R2 are each independently H or a hydrolyzable group, or apharmaceutically acceptable salt thereof.

In an additional embodiment, the invention relates to a method ofactivating at least one cytoprotective kinase (e.g., Akt kinase, IRKkinase, IGF1R kinase, Src kinase) in a cell, comprising contacting thecell with an effective amount of a compound represented by StructuralFormula I:

wherein R1 and R2 are each independently H or a hydrolyzable group, or apharmaceutically acceptable salt thereof. In a particular embodiment,the cytoprotective kinase is Akt kinase, or a kinase that functions inthe same cell signaling pathway as Akt kinase (e.g., IRK kinase).

In another embodiment, the invention relates to a method of treating anischemia or ischemia-reperfusion injury in a mammalian subject,comprising administering to the subject an effective amount of acompound represented by Structural Formula I:

wherein R1 and R2 are each independently H or a hydrolyzable group, or apharmaceutically acceptable salt thereof. In a particular embodiment,the ischemia or ischemia-reperfusion injury is a myocardial ischemia orischemia-reperfusion injury.

In yet another embodiment, the invention relates to a method ofinhibiting apoptosis in a subject, comprising administering to thesubject an effective amount of a compound represented by StructuralFormula I:

wherein R1 and R2 are each independently H or a hydrolyzable group, or apharmaceutically acceptable salt thereof.

In a further embodiment, the invention relates to a method of preventingcytosolic calcium overload in a subject, comprising administering to thesubject an effective amount of a compound represented by StructuralFormula I:

wherein R1 and R2 are each independently H or a hydrolyzable group, or apharmaceutically acceptable salt thereof.

In another embodiment, the invention relates to a method of increasingperoxyl radical absorbance in a subject (e.g., a subject suffering fromischemia), comprising administering to the subject an effective amountof a compound represented by Structural Formula I:

wherein R1 and R2 are each independently H or a hydrolyzable group, or apharmaceutically acceptable salt thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram depicting proposed cell signaling pathwaysaccounting for the potential mechanisms of action for RLip-EA-OH andrelated compounds.

FIG. 2 is a graph depicting the effect of RLip-EA-OH and RLip-OH atvariant concentrations on the level of Akt phosphorylation in A549cells. Data is presented as the mean±sem (N=4) of the backgroundsubtracted ratio of phosphorylated Akt to total Akt.

FIG. 3 is a graph depicting the effect of RLip-EA-OH at variantconcentrations on the level of Akt phosphorylation in A549 cells aloneor in the presence of LY294002, a known phosphotidylinositol-3′-kinaseinhibitor. Data is presented as the mean±sem (N=4) of the backgroundsubtracted ratio of phosphorylated Akt to total Akt.

FIG. 4 is a graph depicting the effect at variant concentrations ofRLip-EA-OH, Ac-EA-OH and RLip-OH on calcium flux in Chinese hamsterovary (CHO) cells. Data is presented as the mean±sem (N=4) measuringcytosolic calcium levels as a percentage of a buffer-only control.

FIG. 5 is a graph depicting the efficacy of RLip-EA-OH in a rat model ofmyocardial ischemia-reperfusion injury. Data is presented as the ratioof myocardial infarct size divided by the total area at risk (MI/AR).The results represent a meta analysis of animals treated via anintracardial injection with RLip-EA-OH at 1 mg/kg (N=64) vs. salinevehicle (N=54). Animals treated with RLip-EA-OH had significantly(p<0.001) reduced (33%) infarct area (dead tissue) to area at risk ratiocompared to those animals receiving saline vehicle.

FIG. 6 is a graph depicting the effect of timing of intracardialadministration of RLip-EA-OH at 1 mg/mL on reduction of myocardialdamage in a rat model of cardiac ischemia-reperfusion injury. Data ispresented as the ratio of myocardial infarct size divided by the totalarea at risk (MI/AR) and displayed as the mean±sem (N=12-15/group).Results indicate that treatment with RLip-EA-OH significantly (p<0.05)reduced myocardial tissue death when administered 15 min pre-occlusion(pre-occlusion, 38%), 15 min after occluding (during occlusion, 24%),and within 1 min after reperfusion (at reperfusion, 32%) compared tothose animals receiving saline vehicle 15 min pre-occlusion.

FIG. 7 is a graph depicting the effect of different doses of RLip-EA-OHin a rat model of myocardial ischemia-reperfusion injury. RLip-EA-OH wasadministered 15 minutes pre-occlusion by intravenous (IV) or intra leftventricular cardiac (IC) administration. Data is presented as the ratioof myocardial infarct size divided by the total area at risk (MI/AR) anddisplayed as the mean±sem (N=10-12/group). Results indicate thattreatment with RLip-EA-OH significantly (p<0.05) reduced myocardialtissue death and was dose dependent.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “alkyl” means a straight or branched hydrocarbon radical having1-10 carbon atoms and includes, for example, methyl, ethyl, n-propyl,isopropyl, n-butyl, sec.-butyl, isobutyl, tert.-butyl, n-pentyl,n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl and the like.

The term “cycloalkyl” means a monocyclic, bicyclic or tricyclic,saturated hydrocarbon ring having 3-10 carbon atoms and includes, forexample, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, bicyclo[2.2.2]octyl, bicyclo[2.2.1]heptyl, spiro[4.4]nonane,adamantyl and the like.

The term “aryl” means an aromatic radical which is a phenyl group, anaphthyl group, an indanyl group or a tetrahydronaphthalene group. Anaryl group is optionally substituted with 1-4 substituents. Exemplarysubstituents include alkyl, alkoxy, alkylthio, alkylsulfonyl, halogen,trifluoromethyl, dialkylamino, nitro, cyano, CO2H, CONH2,N-monoalkyl-substituted amido and N,N-dialkyl-substituted amido.

The term “heteroaryl” means a 5- or 6-membered heteroaromatic radicalwhich may optionally be fused to a saturated or unsaturated ringcontaining 0-4 heteroatoms selected from N, O, and S and includes, forexample, a heteroaromatic radical which is 2- or 3-thienyl, 2- or3-furanyl, 2- or 3-pyrrolyl, 2-, 3-, or 4-pyridyl, 2-pyrazinyl, 2-, 4-,or 5-pyrimidinyl, 3- or 4-pyridazinyl, 1H-indol-6-yl, 1H-indol-5-yl,1H-benzimidazol-6-yl, 1H-benzimidazol-5-yl, 2-, 4-, 5-, 6-, 7- or8-quinazolinyl, 2-, 3-, 5-, 6-, 7- or 8-quinoxalinyl, 2-, 3-, 4-, 5-,6-, 7- or 8-quinolinyl, 1-, 3-, 4-, 5-, 6-, 7- or 8-isoquinolinyl, 2-,4-, or 5-thiazolyl, 2-, 3-, 4-, or 5-pyrazolyl, 2-, 3-, 4-, or5-imidazolyl. A heteroaryl is optionally substituted. Exemplarysubstituents include alkyl, alkoxy, alkylthio, alkylsulfonyl, halogen,trifluoromethyl, dialkylamino, nitro, cyano, CO2H, CONH2,N-monoalkyl-substituted amido and N,N-dialkyl-substituted amido, or byoxo to form an N-oxide.

The term “heterocyclyl” means a 4-, 5-, 6- or 7-membered saturated orpartially unsaturated heterocyclic ring containing 1 to 4 heteroatomsindependently selected from N, O, and S. Exemplary heterocyclyls includepyrrolidine, pyrrolidin-2-one, 1-methylpyrrolidin-2-one, piperidine,piperidin-2-one, 2-pyridone, 4-pyridone, piperazine,1-(2,2,2-trifluoroethyl)piperazine, piperazin-2-one,5,6-dihydropyrimidin-4-one, pyrimidin-4-one, tetrahydrofuran,tetrahydropyran, tetrahydrothiophene, tetrahydrothiopyran,isoxazolidine, 1,3-dioxolane, 1,3-dithiolane, 1,3-dioxane, 1,4-dioxane,1,3-dithiane, 1,4-dithiane, oxazolidin-2-one, imidazolidin-2-one,imidazolidine-2,4-dione, tetrahydropyrimidin-2(1H)-one, morpholine,N-methylmorpholine, morpholin-3-one, 1,3-oxazinan-2-one, thiomorpholine,thiomorpholine 1,1-dioxide, tetrahydro-1,2,5-thiaoxazole 1,1-dioxide,tetrahydro-2H-1,2-thiazine 1,1-dioxide, hexahydro-1,2,6-thiadiazine1,1-dioxide, tetrahydro-1,2,5-thiadiazole 1,1-dioxide andisothiazolidine 1,1-dioxide. A heterocyclyl can be optionallysubstituted with 1-4 substituents. Exemplary substituents include alkyl,haloalkyl and oxo.

Certain of the disclosed compounds may exist in various stereoisomericforms. Stereoisomers are compounds that differ only in their spatialarrangement. Enantiomers are pairs of stereoisomers that arenon-superimposable mirror images of one another, most commonly becausethey contain an asymmetrically substituted carbon atom that acts as achiral center. “Enantiomer” means one of a pair of molecules that aremirror images of each other and are not superimposable. Diastereomersare stereoisomers that are not related as mirror images, most commonlybecause they contain two or more asymmetrically substituted carbonatoms. The symbol “ ” in a structural formula represents the presence ofa chiral carbon center. “R” and “S” represent the configuration ofsubstituents around one or more chiral carbon atoms. Thus, “R*” and “S*”denote the relative configurations of substituents around one or morechiral carbon atoms.

“Racemate” or “racemic mixture” means a compound of equimolar quantitiesof two enantiomers, wherein such mixtures exhibit no optical activity;i.e., they do not rotate the plane of polarized light.

“Levorotatory” signifies that polarized light is rotated to the leftwhen passed through an asymmetric compound. The prefix to designatelevorotary is “1”.

“Dextrorotatory” signifies that polarized light is rotated to the rightwhen passed through an asymmetric compound. The prefix to designatelevorotary is “d”.

“Geometric isomer” means isomers that differ in the orientation ofsubstituent atoms in relationship to a carbon-carbon double bond, to acycloalkyl ring, or to a bridged bicyclic system. Atoms (other thanhydrogen) on each side of a carbon-carbon double bond may be in an E(substituents are on opposite sides of the carbon-carbon double bond) orZ (substituents are oriented on the same side) configuration.

When the stereochemistry of a disclosed compound is named or depicted bystructure, the named or depicted stereoisomer is at least 60%, 70%, 80%,90%, 99% or 99.9% by weight relative to the other stereoisomers. When asingle enantiomer is named or depicted by structure, the depicted ornamed enantiomer is at least 60%, 70%, 80%, 90%, 99% or 99.9% by weightoptically pure. Percent optical purity by weight is the ratio of theweight of the enantiomer over the weight of the enantiomer plus theweight of its optical isomer.

When a disclosed compound has at least one chiral center and is named ordepicted by structure without indicating the stereochemistry, it is tobe understood that the name or structure encompasses one enantiomer ofthe compound free from the corresponding optical isomer, a racemicmixture of the compound and mixtures enriched in one enantiomer relativeto its corresponding optical isomer.

When a disclosed compound has at least two chiral centers and is namedor depicted by structure without indicating the stereochemistry, it isto be understood that the name or structure encompasses a diastereomerfree of other diastereomers, a pair of diastereomers free from otherdiastereomeric pairs, mixtures of diastereomers, mixtures ofdiastereomeric pairs, mixtures of diastereomers in which onediastereomer is enriched relative to the other diastereomer(s) andmixtures of diastereomeric pairs in which one diastereomeric pair isenriched relative to the other diastereomeric pair(s).

In compounds of the invention that contain one or more double bonds, thedesignations “E,” “Z,” “cis,” and “trans” indicate configurationsrelative to the core molecule.

Amino acids may exist in various stereoisomeric forms. The Fischerconvention is commonly used to describe the configuration of the groupsaround the asymmetric carbon atom of an amino acid as compared to thearrangement of the groups around the asymmetric carbon atom ofglyceraldehyde. For α-amino acids, the amino, carboxyl, R (i.e., theside chain) and H groups around the Cα atom correspond to the hydroxyl,aldehyde, CH2OH, and H groups, respectively, of glyceraldehyde:

L-Glyceraldehyde and L-α-amino acids have the same relativeconfiguration and D-glyceraldehyde and D-α-amino acids have the samerelative configuration. The L or D designation does not indicate theamino acid's ability to rotate the plane of polarized light. ManyL-amino acids are dextrorotatory.

As used herein, “substantially pure” means that the depicted or namedcompound is at least about 60% by weight. For example, “substantiallypure” can mean about 60%, 70%, 72%, 75%, 77%, 80%, 82%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.9%, or a percentage between 70% and 100%. In one embodiment,substantially pure means that the depicted or named compound is at leastabout 75%. In a specific embodiment, substantially pure means that thedepicted or named compound is at least about 90% by weight. Thesubstantially pure composition comprising a compound represented byStructural Formula I can comprise the four compounds represented byStructural Formulae IV, V, VI or VII, either alone, or in anycombination thereof.

As used herein, an “effective amount” is an amount sufficient to achievea desired effect under the conditions of administration, in vitro, invivo or ex vivo, such as, for example, an amount sufficient to activateone or more cytoprotective kinases in a cell, an amount sufficient toinhibit apoptosis of a cell and an amount sufficient to inhibit (e.g.,prevent, delay) ischemia and ischemia reperfusion injury (e.g., in asubject). The effectiveness of a therapy can be determined by suitablemethods known by those of skill in the art including those describedherein.

As defined herein, a “therapeutically effective amount” is an amountsufficient to achieve a desired therapeutic or prophylactic effect in asubject in need thereof under the conditions of administration, such as,for example, an amount sufficient to inhibit (e.g., prevent, delay)ischemia and ischemia reperfusion injury in a subject (e.g., byinhibiting apoptosis of one or more affected cells in the subject). Theeffectiveness of a therapy can be determined by suitable methods knownby those of skill in the art.

The present invention is based, in part, on Applicants' discovery thatthe lipoic acid derivative compounds described herein havecytoprotective and anti-oxidative properties. In particular, Applicantshave shown that a certain lipoic acid derivative, RLip-EA-OH, activatesAkt kinase and other kinases (e.g., IRK, IGF1R, Src) that are known tomediate cell signaling pathways that inhibit apoptosis and promote cellsurvival (FIG. 1). Applicants have further shown that RLip-EA-OH canreduce the extent of ischemia and ischemia-reperfusion injury in ananimal model of myocardial ischemia-reperfusion injury.

In one embodiment, the invention relates to compositions comprising asubstantially pure compound represented by Structural Formula I:

wherein R1 and R2 are each independently H or a hydrolyzable group, or apharmaceutically acceptable salt thereof.

As used herein, the term “hydrolyzable group” refers to a moiety that,when present in a molecule of the invention, yields a carboxylic acid orsalt thereof upon hydrolysis. Hydrolysis can occur, for example,spontaneously under acidic or basic conditions in a physiologicalenvironment (e.g., blood, metabolically active tissues such as, forexample, liver, kidney, lung, brain), or can be catalyzed by anenzyme(s), (e.g., esterase, peptidases, hydrolases, oxidases,dehydrogenases, lyases or ligases). A hydrolyzable group can confer upona compound of the invention advantageous properties in vivo, such asimproved water solubility, improved circulating half-life in the blood,improved uptake, improved duration of action, or improved onset ofaction.

In one embodiment, the hydrolyzable group does not destroy thebiological activity of the compound. In an alternative embodiment, acompound with a hydrolyzable group can be biologically inactive, but canbe converted in vivo to a biologically active compound.

Compounds of the invention that include hydrolyzable groups may act asprodrugs. As used herein, the term “prodrug” means a compound that canbe hydrolyzed, oxidized, metabolized or otherwise react under biologicalconditions to provide a compound of the invention. Prodrugs may becomeactive upon such reaction under biological conditions, or they may haveactivity in their unreacted forms. A prodrug may undergo reducedmetabolism under physiological conditions (e.g., due to the presence ofa hydrolyzable group), thereby resulting in improved circulatinghalf-life of the prodrug (e.g., in the blood). Prodrugs can typically beprepared using well-known methods, such as those described by Burger'sMedicinal Chemistry and Drug Discovery (1995) 172-178, 949-982 (ManfredE. Wolff ed., 5th ed).

In one embodiment, the hydrolyzable group is selected from the groupconsisting of (C1-C10)alkyl, (C2-C10)alkenyl, (C2-C10)alkynyl,(C1-C10)alkoxy(C1-C10)alkyl, (C1-C10)alkoxy(C1-C10)alkoxy(C1-C10)alkyl,aryl and aryl(C1-C10)alkyl, wherein each is optionally substituted with1 to 3 substituents selected from the group consisting of halo, nitro,cyano, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, amino,(C1-C6)alkylamino, di(C1-C6)alkylamino, (C1-C6)alkyl, halo(C1-C6)alkyl,(C1-C6)alkoxy, halo(C1-C6)alkoxy, morpholino, phenyl, and benzyl.

In another embodiment, the hydrolyzable group is selected from the groupconsisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,isobutyl, tert-butyl, pentyl, hexyl, heptyl, allyl, ethoxymethyl,methoxyethyl, methoxyethoxymethyl, methoxyethoxyethyl, benzyl,pentafluorophenyl, 2-N-(morpoholino)ethyl, dimethylaminoethyl andpara-methoxybenzyl.

Hydrolysis of the hydrolyzable group generates a carboxylic acid. Forexample, the tert.-butyl in Compound A is cleaved to generate thecarboxylic acid groups in Compound B in mildly acidic conditions:

R1 and R2 may be different hydrolyzable groups, resulting in compoundssuch as Compound C, where two different esters are present. Use ofdifferent hydrolyzable groups can allow for selective hydrolysis of aparticular ester. For example, either R1 or R2 can be a hydrolyzablegroup stable to acidic environments and the other can be a hydrolyzablegroup stable to basic environments. In an alternative embodiment, eitherR1 or R2 can be a hydrolyzable group cleaved by a particular enzyme,while the other is not cleaved by that enzyme. In some embodiments, thehydrolysis of the two esters may occur simultaneously. Alternatively,the hydrolysis of the two esters may be step-wise. In another example,the tert.-butyl group in Compound C is cleaved under mildly acidicconditions while the 2-N-morpolinoethyl moiety may be enzymaticallycleaved with the lipase from R. niveus:

Methods for the selection, introduction and subsequent removal ofhydrolyzable groups are well known to those skilled in the art. (T. W.Greene and P. G. M. Wuts “Protective Groups in Organic Synthesis” JohnWiley & Sons, Inc., New York 1999).

Alternatively, only one of R1 or R2 may be present, resulting in ahydrolysis of a single ester to generate the carboxylic acid or saltthereof:

One skilled in the art will understand that other hydrolyzableprotecting groups can be employed with the compounds of the presentinvention to obtain prodrugs encompassed by the present description.

The compounds of the invention may be present in the form ofpharmaceutically acceptable salts. For use in medicines, the salts ofthe compounds of the invention refer to non-toxic pharmaceuticallyacceptable salts.

The pharmaceutically acceptable salts of the disclosed compounds includeacid addition salts and base addition salts. The term “pharmaceuticallyacceptable salts” embraces salts commonly used to form alkali metalsalts and to form addition salts of free acids or free bases. The natureof the salt is not critical, provided that it is pharmaceuticallyacceptable.

Suitable pharmaceutically acceptable acid addition salts of thedisclosed compounds may be prepared from an inorganic acid or an organicacid. Examples of such inorganic acids are hydrochloric, hydrobromic,hydroiodic, nitric, carbonic, sulfuric and phosphoric acid. Appropriateorganic acids may be selected from aliphatic, cycloaliphatic, aromatic,arylaliphatic, heterocyclic, carboxylic and sulfonic classes of organicacids, examples of which are formic, acetic, propionic, succinic,glycolic, gluconic, maleic, embonic (pamoic), methanesulfonic,ethanesulfonic, 2-hydroxyethanesulfonic, pantothenic, benzenesulfonic,toluenesulfonic, sulfanilic, mesylic, cyclohexylaminosulfonic, stearic,algenic, β-hydroxybutyric, malonic, galactic, and galacturonic acid.Pharmaceutically acceptable acidic/anionic salts also include, theacetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide,calcium edetate, camsylate, carbonate, chloride, citrate,dihydrochloride, edetate, edisylate, estolate, esylate, fumarate,glyceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate,hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate,lactate, lactobionate, malate, maleate, malonate, mandelate, mesylate,methylsulfate, mucate, napsylate, nitrate, pamoate, pantothenate,phosphate/diphospate, polygalacturonate, salicylate, stearate,subacetate, succinate, sulfate, hydrogensulfate, tannate, tartrate,teoclate, tosylate, and triethiodide salts.

Suitable pharmaceutically acceptable base addition salts of thedisclosed compounds include, but are not limited to, metallic salts madefrom aluminum, calcium, lithium, magnesium, potassium, sodium and zincor organic salts made from N,N′-dibenzylethylene-diamine,chloroprocaine, choline, diethanolamine, ethylenediamine,N-methylglucamine, lysine, arginine and procaine. All of these salts maybe prepared by conventional means from the corresponding compoundrepresented by the disclosed compound by treating, for example, thedisclosed compounds with the appropriate acid or base. Pharmaceuticallyacceptable basic/cationic salts also include, the diethanolamine,ammonium, ethanolamine, piperazine and triethanolamine salts.

In an embodiment, the pharmaceutically acceptable salt comprises amonovalent cation or a divalent cation. In a particular embodiment, thepharmaceutically acceptable salt is a lysine salt.

In another embodiment, the monovalent cation is a monovalent metalcation and the divalent cation is a divalent metal cation. In aparticular embodiment, the monovalent metal cation is a sodium cation.

In another embodiment, the invention relates to compositions comprisinga substantially pure compound represented by Structural Formula II:

or a pharmaceutically acceptable salt thereof. As used herein,RLip-EA-OH refers to Structural Formula II.

In a further embodiment, the invention relates to compositionscomprising a substantially pure compound represented by StructuralFormula III:

In another embodiment, the invention relates to compositions comprisinga substantially pure compound represented by Structural Formula IV:

or a pharmaceutically acceptable salt thereof. The values of R1 and R2are as defined above for Structural Formula (I).

In another embodiment, the invention relates to compositions comprisinga substantially pure compound represented by Structural Formula V:

or a pharmaceutically acceptable salt thereof. The values of R1 and R2are as defined above for Structural Formula (I).

In another embodiment, the invention relates to compositions comprisinga substantially pure compound represented by Structural Formula VI:

or a pharmaceutically acceptable salt thereof. The values of R1 and R2are as defined above for Structural Formula (I).

In another embodiment, the invention relates to compositions comprisinga substantially pure compound represented by Structural Formula VII:

or a pharmaceutically acceptable salt thereof. The values of R1 and R2are as defined above for Structural Formula (I).

A composition of the invention may, alternatively or in addition to thedisclosed compounds, comprise a pharmaceutically acceptable salt of acompound represented by the disclosed compounds, or a prodrug orpharmaceutically active metabolite of such a compound or salt and one ormore pharmaceutically acceptable carriers and are delivered to arecipient subject (preferably a human) in accordance with known methodsof drug delivery. The compounds of the present invention may beadministered alone or in combination with at least one other agent knownor believed by the applicants to be useful for the activation ofcytoprotective kinases and/or the treatment of ischemia orischemia-reperfusion injuries.

Alternatively, a composition of the invention may comprise a compoundrepresented by the disclosed compounds or a pharmaceutical salt thereofas the only pharmaceutically active agent in the composition.

In another embodiment, the invention relates to a compositioncomprising: i) a pharmaceutically acceptable carrier or diluent; and ii)a compound represented by Structural Formula I:

wherein R1 and R2 are each independently H or a hydrolyzable group, or apharmaceutically acceptable salt thereof.

The pharmaceutically acceptable compositions of the present inventioncomprise one or more compounds disclosed herein in association with oneor more nontoxic, pharmaceutically acceptable carriers and/or diluentsand/or adjuvants and/or excipients, collectively referred to herein as“carrier” materials, and, if desired, other active ingredients.

The present invention also relates to methods of activating acytoprotective kinase in a cell. As used herein, the term“cytoprotective kinase” refers to a kinase that, when activated,phosphorylates components of one or more cell signaling pathways thatpromote cell survival and/or inhibit cell death (e.g., apoptosis).

Accordingly, in one embodiment, the invention relates to a method ofactivating a cytoprotective kinase (e.g., insulin receptor kinase, Aktkinase, insulin-like growth factor 1 receptor kinase, Src kinase) in acell, comprising contacting the cell with an effective amount of acompound represented by Structural Formula I:

wherein R1 and R2 are each independently H or a hydrolyzable group, or apharmaceutically acceptable salt thereof.

In a particular embodiment, the invention relates to a method ofactivating a kinase whose pathway is cytoprotective in a cell,comprising contacting the cell with an effective amount of a compoundrepresented by Structural Formula II:

or a pharmaceutically acceptable salt thereof.

In another embodiment, the invention relates to a method of activating acytoprotective kinase in a cell, comprising contacting the cell with aneffective amount of a compound represented by Structural Formula III:

Activation of a cytoprotective kinase can lead to activation of one ormore cytoprotective cell signaling pathways that include thecytoprotective kinase. In a particular embodiment, activation of one ormore cytoprotective kinases in a cell by the compounds of the inventioncan inhibit (e.g., prevent, delay) apoptosis of the cell in which thekinase(s) has been activated.

The methods of the invention relating to activation of cytoprotectivekinases can be performed in vitro (e.g., using cultured cells, usingisolated cells) or in vivo (e.g., by administering a compound(s) of theinvention to a living organism). In a particular embodiment, thecompounds of the invention are used in a method to activate one or morecytoprotective kinases in one or more cells in a human.

In one embodiment, the cytoprotective kinase is Akt kinase. Activationof Akt kinase can lead to activation of one or more Akt cell signalingpathways that are cytoprotective. Akt kinase, also known as Akt, PKB andRac-PK, belongs to the Akt/PKB family of serine/threonine kinases andhas been shown to be involved in many diverse signaling pathways(Alessi, and Cohen, Curr. Opin. Genet. Dev. 8 (1998), 55-62) includingpathways related to cell survival and proliferation (Song, G., Ouyang,G., and Bao, S., The activation of Akt/PKB signaling pathway and cellsurvival. J Cell Mol Med 2005 9:59; Hausenloy, D. J., Yellon, D. M.,Reperfusion injury salvage kinase signaling: taking a RISK forcardioprotection. Heart Fail Rev 2007, 12:217.). Akt consists of anN-terminal lipid-binding pleckstrin-homology domain and a C-terminalcatalytic domain. In resting cells, all Akt isoforms reside in thecytoplasm but translocate to the plasma membrane following stimulationwith external ligands. Translocation and subsequent activation isinduced by several different ligands including PDGF, IGF, EGF, βFGF andinsulin. This activation depends on PI3-kinase activity and requireshierarchial phosphorylation of Thr308 and Ser473 of Akt by PDK-1 andPDK-2, respectively (Alessi et al., Curr. Biol. 8 (1998), 69-81). Onceactivated, Akt mediates several different functions, includingprevention of apoptosis, induction of differentiation and/orproliferation, protein synthesis and the metabolic effects of insulin.

As described in Example 2 herein, compounds of the invention increaseAkt phosphorylation in a cell-based in vitro assay. Akt kinasephosphorylation in a cell can be assessed using one or more in vitro Aktkinase phosphorylation assays known in the art including, for example,kits and assays for testing AKT phosphorylation in cells available fromcommercial suppliers (e.g., Cellomics Phospho-AKT Activation Kit, ThermoScientific; Akt Activity Assay Kit, BioVision Incorporated; FACE™ AKTin-cell Western analysis for phospho AKT (S473), Active Motif; PathScan®Phospho-Akt (Thr308) Sandwich ELISA Kit, Cell Signaling Technology;AlphaScreen® SureFire® Phospho-AKT Assay Kits, Perkin Elmer; AktActivity Immunoassay Kit, EMD Biosciences). An exemplary assay forassessing Akt kinase phosphorylation is described herein in Example 2.(Chen, H., Kovar, J., Sissons, S., et. al. A cell basedimmunocytochemical assay for monitoring kinase signaling pathways anddrug efficacy. Analyt Biochem 2005 338: 136)

Akt activation can also be assessed in vivo, e.g., by immunodetectionmethods performed on a cell sample obtained from a subject. SeveralAkt-specific antibodies, including phospho-specific Akt antibodies(e.g., specific for phospho-Ser473, specific for phospho-Thr308), arecommercially available (e.g., Perkin Elmer).

In another embodiment, the cytoprotective kinase is insulin receptorkinase (“IRK”) (Diesel, B., Kulhanek-Heinze, S., Holtje, M., et. al.,αα-Lipoic Acid as a directly binding activator of the insulin receptor:protection from hepatocyte apoptosis. Biochemistry, 2007 46:2146;Hausenloy, D. J., Yellon, D. M. New directions for protecting the heartagainst ischemia-reperfusion injury: targeting the reperfusion injurysalvage kinase (RISK)-pathway. Cardiovasc Res 2004 61:448). IRKactivation leads to phosphorylation and activation of Akt (Alessi, D.R., Andjelkovic, M., Caudwell, B., Cron, P., Morrice, N., Cohen, P.,Hemmings, B. A. Mechanism of activation of protein kinase B by insulinand IGF-1. EMBO J 1996 15:6541-6551). Activation of IRK can lead toactivation of one or more IRK cell signaling pathways that arecytoprotective. As described in Example 3 herein, compounds of theinvention activate IRK in a biochemical assay in vitro. IRK activationcan be assessed using one or more in vitro IRK activation assays knownin the art. An exemplary assay for assessing IRK activation is describedherein in Example 3. (Mobility Shift Kinase Assay, Caliper LifeSciences, Hanover, Md.)

In another embodiment, the cytoprotective kinase is insulin-like growthfactor 1 receptor (“IGF1R”) kinase. Activation of IGF1R kinase can leadto activation of one or more IGF1R cell signaling pathways that arecytoprotective. As described in Example 4 herein, compounds of theinvention activate IGF1R kinase in a biochemical assay in vitro. IGF1Rkinase activation can be assessed using one or more in vitro IGF1Rkinase activation assays known in the art. An exemplary assay forassessing IGF1R kinase activation is described herein in Example 4.

In a further embodiment, the cytoprotective kinase is Src kinase.Activation of Src kinase can lead to activation of one or more Src cellsignaling pathways that are cytoprotective. As described in Example 4herein, compounds of the invention activate Src kinase in a biochemicalassay in vitro. Src kinase activation can be assessed using one or morein vitro Src kinase activation assays known in the art.

IGF1R and Src tyrosine kinases play a role in protecting the heart fromischemia-reperfusion injury (Buddhadeb, D., Takano, H., Tang, X.-L., etal. Role of Src protein tyrosine kinase in late preconditioning againstmyocardial infarction. Am J Physiol 2002 283:H549; Pasdois, P., Quinlan,C. L., Rissa, A., et al. Ouabain protects rat hearts againstischemia-reperfusion injury via pathway involving Src kinase, mitoKATP,and ROS. Am J Physiol 2006, 292:H1470; Suzuki, Y. J. Growth factorsignaling for cardioprotection against oxidative stress-inducedapoptosis. Antiox Redox Signal 2003, 5:741; Hausenloy, D. J., Yellon, D.M., New directions for protecting the heart againstischaemia-reperfusion injury: Targeting the Reperfusion Injury SalvageKinase (RISK)-pathway. Cardiovasc Res 2004 61:448).

According to the invention, activation of one or more cytoprotectivekinases in a cell by the compounds of the invention can inhibit (e.g.,prevent, delay) apoptosis of the cell. Methods of assessing apoptosisare well known in the art. Microscopic analysis (e.g., light microscopy,electron microscopy, confocal microscopy, laser-scanning microscopy) forvisualizing apoptotic cells (e.g., by detecting morphological changesassociated with apoptosis, such as chromatin condensation andcytoplasmic shrinking) is typically employed to study apoptotic cells.

The study of DNA fragmentation in agarose gels is also considered to beindicative of apoptosis. A number of techniques take advantage of DNAfragmentation for labeling the fragments and thus for quantifying theproportion of apoptotic cells. Each DNA fragment has a 3′-OH terminalportion. This terminal fragment can be labeled in various ways (forinstance, with the help of a modified terminal deoxynucleotidyltransferase), so that the labeling rate is proportional to the degree ofDNA fragmentation.

In particular, TdT-mediated dUTP Nick-End Labeling, or TUNEL, is atechnique for detecting fragmented DNA, which occurs near the final stepin the apoptotic process. Fragmented DNA of apoptotic cells canincorporate fluorescein-dUTP at 3′-OH at DNA ends using the enzymeTerminal Deoxynucleotidyl Transferase (TdT), which forms a polymerictail using the principle of the TUNEL assay. The labeled DNA can then bevisualized directly by fluorescence microscopy or quantitated by flowcytometry.

Some current techniques take advantage of the changes in membranephospholipids that occur early in apoptotic cells. The negativelycharged membrane phospholipids exposed to the external environment bythe apoptotic cell are labeled with fluorochrome-conjugated molecules,and the percentage of fluorescent cells can be easily quantified.

Apoptosis can also be detected using fluorescently-conjugated Annexin V.Annexin V is an anticoagulant protein that preferentially bindsnegatively charged phospholipids. An early step in the apoptotic processis disruption of membrane phospholipid asymmetry, exposingphosphatidylserine (PS) on the outer leaflet of the cytoplasmicmembrane. Fluorescently conjugated Annexin V can be used to detect thisexternalization of phosphatidylserine on intact living cells. Propidiumiodide is often combined as a second flurochrome to detect necroticcells. Induction of apoptosis leads to procaspase-3 proteolytic cleavageto generate an active 18 kDa caspase-3 fragment which then targets keymodulators of the apoptotic pathway including poly-ADP-ribose polymeraseand other caspases, for cleavage. Assays for detecting other activecaspases in apoptotic cells are known in the art (e.g., Caspase-Glo®Assays, Promega).

Apoptotic cells can also be detected using the active 18 kDa caspase-3fragment as a marker. Induction of apoptosis leads to procaspase-3proteolytic cleavage to generate an active 18 kDa caspase-3 fragmentwhich then targets key modulators of the apoptotic pathway, includingpoly-ADP-ribose polymerase and other caspases, for cleavage. Severalantibodies that recognize only the active 18 kDa fragment are availablefrom commercial suppliers (e.g., BD Biosciences, Chemicon, CellSignaling Technology, Trevigen).

In addition, flow cytometry assays can be employed to monitor andquantify nuclear changes associated with apoptotic cells.

An exemplary assay for detecting inhibition of apoptosis is describedherein in Example 5.

The activation of cellular cytoprotective kinases also have utility inthe treatment of conditions resulting from excess or unwanted apoptoticcell death in an affected tissue or organ, leading to damage anddysfunction. Such conditions include, inter alia, ischemia andischemia-reperfusion injury. Accordingly, the invention also relates tomethods of treating an ischemia or ischemia-reperfusion injury in amammalian subject, comprising administering to the subject an effectiveamount of a compound represented by Structural Formula I:

wherein R1 and R2 are each independently H or a hydrolyzable group, or apharmaceutically acceptable salt thereof.

In a particular embodiment, the invention relates to a method oftreating an ischemia or ischemia-reperfusion injury in a mammaliansubject, comprising administering to the subject an effective amount ofa compound represented by Structural Formula II:

or a pharmaceutically acceptable salt thereof.

In another embodiment, the invention relates to a method of treating anischemia or ischemia-reperfusion injury in a mammalian subject,comprising administering to the subject an effective amount of acompound represented by Structural Formula III:

As used herein, the “injury resulting from ischemia,” “injury caused byischemia” and “ischemic injury” refer to an injury to a cell, tissue ororgan caused by ischemia, or an insufficient supply of blood (e.g., dueto a blocked artery), and, thus, oxygen, resulting in damage ordysfunction of the tissue or organ (Piper, H. M., Abdallah, C., Schafer,C., Annals of Thoracic Surgery 2003, 75:644; Yellon, D. M., Hausenloy,D. J., New England Journal of Medicine 2007, 357:1121). Injuries thatresult from ischemia can affect various tissues and organs. Suchinjuries may be treated by the compounds and methods of the invention,including, for example, injuries caused by cardiovascular ischemia,cerebrovascular ischemia, renal ischemia, hepatic ischemia, ischemiccardiomyopathy, cutaneous ischemia, bowel ischemia, intestinal ischemia,gastric ischemia, pulmonary ischemia, pancreatic ischemia, skeletalmuscle ischemia, abdominal muscle ischemia, limb ischemia, ischemiccolitis, mesenteric ischemia and silent ischemia. Thus, an injuryresulting from ischemia can affect, for example, a heart, kidney, liver,brain, muscle, intestine, stomach, lung or skin.

In a particular embodiment, the injury resulting from ischemia is theresult of a myocardial ischemia. An injury resulting from a myocardialischemia can result from, for example, a myocardial infarction (e.g., anacute myocardial infarction) in an individual.

In another embodiment, the injury resulting from ischemia is an injuryresulting from cerebral ischemia (e.g., a stroke) in an individual.

In another embodiment, the injury resulting from ischemia is anischemia-reperfusion injury. As used herein, the term“ischemia-reperfusion injury” refers to an injury resulting from therestoration of blood flow to an area of a tissue or organ that hadpreviously experienced deficient blood flow due to an ischemic event.Oxidative stresses associated with reperfusion may cause damage to theaffected tissues or organs. Ischemia-reperfusion injury is characterizedbiochemically by a depletion of oxygen during an ischemic event followedby reoxygenation and the concomitant generation of reactive oxygenspecies during reperfusion (Piper, H. M., Abdallah, C., Schafer, C.,Annals of Thoracic Surgery 2003, 75:644; Yellon, D. M., Hausenloy, D.J., New England Journal of Medicine 2007, 357:1121).

An ischemia-reperfusion injury can be caused, for example, by a naturalevent (e.g., restoration of blood flow following a myocardialinfarction), a trauma, or by one or more surgical procedures or othertherapeutic interventions that restore blood flow to a tissue or organthat has been subjected to a diminished supply of blood. Such surgicalprocedures include, for example, coronary artery bypass graft surgery,coronary angioplasty, organ transplant surgery and the like. In aparticular embodiment the compounds and methods of the invention areuseful for treating peri-operative cardiac damage caused by an ischemiaor ischemia-reperfusion injury.

For the treatment of ischemic and ischemia-reperfusion injuries causedby therapeutic interventions, such as surgical procedures, it ispreferable that a compound of the invention is administered to a subjectundergoing treatment prior to the therapeutic intervention (e.g.,cardiac surgery, organ transplant). For example, a compound of theinvention can be administered to a subject undergoing treatment, e.g.,about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5hours, about 12 hours, about 24 hours, or about 48 hours prior to thetherapeutic intervention. A compound of the invention can also beadministered to a subject undergoing treatment, for example, about 5minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30minutes or about 45 minutes prior to the therapeutic intervention.

Alternatively, or in addition, a compound of the invention can beadministered to a subject undergoing treatment at the time of, orduring, the therapeutic intervention. For example, the compound can beadministered one or more times during the course of a therapeuticintervention in intervals (e.g., 15 minute intervals). Alternatively, acompound can be administered continuously throughout the duration of atherapeutic intervention.

Furthermore, a compound of the invention can be administered to asubject undergoing treatment after a therapeutic intervention. Forexample, a compound of the invention can be administered to a subjectundergoing treatment, e.g., about 1 hour, about 2 hours, about 3 hours,about 4 hours, about 5 hours, about 12 hours, about 24 hours, or about48 hours after the therapeutic intervention. A compound of the inventioncan also be administered to a subject undergoing treatment, for example,about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes,about 30 minutes or about 45 minutes after the therapeutic intervention.

A compound of the invention can also be used to inhibit an ischemia orischemia-reperfusion injury to a cell, tissue or organ, ex vivo, priorto a therapeutic intervention (e.g., a tissue employed in a graftprocedure, an organ employed in an organ transplant surgery). Forexample, prior to transplant of an organ into a host individual (e.g.,during storage or transport of the organ in a sterile environment), theorgan can be contacted with a compound of the invention (e.g., bathed ina solution comprising a compound of the invention) to inhibit ischemiaor ischemia-reperfusion injury.

As described herein, conditions resulting from ischemia, and injuriescaused by ischemia or ischemia-reperfusion, can induce apoptotic celldeath in an affected cell, tissue or organ, leading to damage anddysfunction. Accordingly, the compounds of the invention also haveutility in methods of inhibiting apoptosis in a cell, a tissue or anorgan (e.g., a transplant tissue or organ or a cell, tissue or organ ina subject), wherein the cell, tissue or organ has experienced anischemia or other condition or disorder that results in excessive orunwanted apoptosis. The methods comprise contacting the cells, tissue,or organ with, or administering to the subject, an effective amount of acompound represented by Structural Formula I:

wherein R1 and R2 are each independently H or a hydrolyzable group, or apharmaceutically acceptable salt thereof.

In a particular embodiment, the invention relates to a method ofinhibiting apoptosis in a cell, tissue or organ, wherein the cell,tissue or organ has experienced an ischemia or other condition ordisorder that results in excessive or unwanted apoptosis, comprisingadministering to the subject an effective amount of a compoundrepresented by Structural Formula II:

or a pharmaceutically acceptable salt thereof.

In another embodiment, the invention relates to a method of inhibitingapoptosis in a cell, tissue or organ, wherein the cell, tissue or organhas experienced an ischemia or other condition or disorder that resultsin excessive or unwanted apoptosis, comprising administering to thesubject an effective amount of a compound represented Structural FormulaIII:

Methods for assessing apoptosis in cells, tissues or organs are known inthe art and include those described herein.

Conditions associated with unwanted and/or excess apoptosis that aretreatable by the compounds and methods of the invention include, but arenot limited to, neurodegenerative diseases associated with excessapoptosis (e.g., Parkinson's Disease, Alzheimer's Disease, amyotrophiclateral sclerosis, retinitis pigmentosa, epilepsy), haematologicdiseases associated with excess apoptosis (e.g., aplastic anaemia,myelodysplastic syndrome, T CD4+ lymphocytopenia, G6PD deficiency),tissue damage associated with excess apopotosis (e.g., myocardialinfarction, cerebrovascular accident, ischemic renal damage, polycystickidney disease), AIDS, and preeclampsia.

One of the hallmarks of ischemia-reperfusion injury is an increase incytosolic calcium levels, resulting from a depletion of oxygen during anischemic event (Piper, H. M., Abdallah, C., Schafer, C., Annals ofThoracic Surgery 2003, 75:644; Yellon, D. M., Hausenloy, D. J., NewEngland Journal of Medicine 2007, 357:1121). It has been postulated thatthe increase in cytosolic calcium combined with an increase in freeradicals triggers apoptosis (Chen, X., Zhang, X., Hubo, H., et al., CircRes 2005, 97:1009; Lopes-Neblina, F., Toledo, A. H., Toledu-Pereyra, L.H. J Invest Surg 2005, 18:335). However, to date, treatments of patientswith acute myocardial infarction with either an antagonist to block theinflux of calcium or with a scavenger of the reactive oxygen species haseach yielded disappointing clinical outcomes (Yellon, D. M., Hausenloy,D. J., New England Journal of Medicine 2007, 357:1121).

In addition, through pro-survival pathways activated by Akt, cytosoliccalcium overload is inhibited (Joseph, S. K., Hajnoczky, G., Apoptosis2007, 12:951; Pinton, P., Rizzuto, R., Cell Death Diff 2006, 13:1409;Khan, M. T., Wagner, L. II, Yule, D. I., Bhanumathy, C., Joseph, S. K.2006, Akt kinase phosphorylation of inositol 1,4,5-triphosphatereceptors. J Biol Chem 281:3731). The Akt dependent signaling pathwayalso prevents intracellular calcium overload by regulation of Bc1-2(Raphael, J., Abedat, S., Rivo, J., et al., J Pharmacol Exp Ther 2006,318:186; Thomenius, M. J. and Distelhorst, C. W., J Cell Sci 2003,116:4493).

Accordingly, the compounds of the invention, which induce Aktactivation, also have utility in methods of decreasing cytosolic calciumin a cell, tissue or organ (e.g., in a subject suffering from anischemia). The methods comprise administering to the subject aneffective amount of a compound represented by Structural Formula I:

wherein R1 and R2 are each independently H or a hydrolyzable group, or apharmaceutically acceptable salt thereof.

In a particular embodiment, the invention relates to a method ofdecreasing cytosolic calcium in a cell, tissue or organ, comprisingadministering to the subject an effective amount of a compoundrepresented by Structural Formula II:

or a pharmaceutically acceptable salt thereof.

In another embodiment, the invention relates to a method of decreasingcytosolic calcium in a cell, tissue or organ, comprising administeringto the subject an effective amount of a compound represented StructuralFormula III:

An exemplary assay for detecting levels of cytosolic calcium isdescribed herein in Example 6.

Compounds of the invention also display an enhanced capacity for peroxylradical absorbance. Biological organisms generate harmful reactiveoxygen species (ROS) and various free radicals in the course of normalmetabolic activities of tissues such as brain, heart, lung, and muscletissue (Halliwell, B. and Gutteridge, J. M. C., eds. (Oxford: ClarendonPress, 1989)). Recognition of the role of ROS and free radicals in avariety of important diseases and drug side effects has grownappreciably over recent years. Many studies have demonstrated that alarge number of disease states and harmful side effects of therapeuticdrugs are linked with a failure of the antioxidant defense system of anindividual to keep up with the rate of generation of ROS and variousfree radicals (see, for example, Chan, et al., Adv. Neurol., 1996,71:271-279; DiGuiseppi, J. and Fridovich, I., Crit. Rev. Toxicol., 1984,12:315-342). For example, abnormally high ROS levels have been foundunder conditions of anoxia elicited by ischemia during a stroke oranoxia generated in heart muscle during myocardial infarction (see, forexample, Walton, M. et al., Brain Res. Rev., 1999, 29:137-168;Pulsinelli, W. A. et al., Ann. Neurol., 1982, 11: 499-502; Lucchesi, B.R., Am. J. Cardiol., 1990, 65:141-231). In addition, an elevation of ROSand free radicals has also been linked with reperfusion damage afterrenal transplants.

Accordingly, an elevation of ROS and free radicals has been linked withthe progression and complications developed in many diseases, drugtreatments, traumas, and degenerative conditions including oxidativestress induced damage with age, Tardive dyskinesia, Parkinson's disease,Huntington's disease, degenerative eye diseases, septic shock, head andspinal cord injuries, Alzheimer's disease, ulcerative colitis, humanleukemia and other cancers, and diabetes (see, for example, Ratanis,Pharmaceutical Executive, pp. 74-80 (April 1991)).

For example, elevated levels of ROS and free radicals are known to begenerated in cells and tissues during reperfusion after an ischemicevent. Such increased levels of ROS and free radicals can causeconsiderable damage to an already stressed or debilitated organ ortissue. The compounds of this invention, which display peroxyl radicalabsorbance capacity, may be used to treat high levels of harmful freeradicals present after reperfusion injuries that occur in diseases andconditions such as stroke, heart attack, or renal disease and kidneytransplants. If the ischemic event has already occurred, as in strokeand heart attack, a compound described herein may be administered to theindividual to detoxify the elevated ROS and free radicals alreadypresent in the blood and affected tissue or organ.

Alternatively, if the ischemic event is anticipated as in organtransplantation, then compounds described herein may be administeredprophylactically, prior to the operation or ischemic event.

The compounds described herein may be used to treat any disease orcondition associated with undesirable levels of ROS and free radicals,or to prevent any disease, disorder or condition caused by undesirablelevels of ROS and free radicals. According to the invention, thecompounds described herein may also be administered to provide atherapeutic or prophylactic treatment of elevated ROS and other freeradicals associated with a variety of other diseases and conditions,including, but not limited to, oxygen toxicity in premature infants,burns and physical trauma to tissues and organs, septic shock,polytraumatous shock, head trauma, brain trauma, spinal cord injuries,Parkinson's disease, amyotrophic lateral sclerosis (ALS), Alzheimer'sdisease, age-related elevation of ROS and free radicals, senility,ulcerative colitis, human leukemia and other cancers, Down syndrome,arthritis, macular degeneration, schizophrenia, epilepsy, radiationdamage (including UV-induced skin damage), and drug-induced increase inROS and free radicals.

A progressive rise of oxidative stress due to the formation of ROS andfree radicals also occurs during aging (see, e.g., Mecocci, P. et al.,Free Radio. Biol. Med., 2000, 28: 1243-1248). This has been detected byfinding an increase in the formation of lipid peroxidates in rat tissues(Erdincler, D. S., et al., Clin. Chim. Acta, 1997, 265: 77-84) and bloodcells in elderly human patients (Congi, F., et al., Presse. Med., 1995,24: 1115-1118). Accordingly, the compounds described herein, which areable to absorb peroxyl radicals, are also well suited for use in methodsof preventing and/or counteracting increased tissue damage and decreasedlife expectancy due to elevated levels of ROS and free radicals thataccompany the aging process.

Thus, the compounds of the invention have utility in the treatment ofconditions and disorders caused by harmful reactive oxygen species (ROS)and other free radicals. Accordingly, the invention further relates tomethods of increasing peroxyl radical absorbance in a tissue in asubject (e.g., a subject suffering from an ischemia), comprisingadministering to the subject an effective amount of a compoundrepresented by Structural Formula I:

wherein R1 and R2 are each independently H or a hydrolyzable group, or apharmaceutically acceptable salt thereof.

In a particular embodiment, the invention relates to a method ofincreasing peroxyl radical absorbance in a tissue in a subject,comprising administering to the subject an effective amount of acompound represented by Structural Formula II:

or a pharmaceutically acceptable salt thereof.

In another embodiment, the invention relates to a method of increasingperoxyl radical absorbance in a tissue in a subject, comprisingadministering to the subject an effective amount of a compoundrepresented Structural Formula III:

An exemplary assay for detecting peroxyl radical absorbance is describedherein in Example 7. Other methods of detecting free radical absorbanceare described in U.S. Pat. No. 6,890,896, the contents of which areincorporated herein by reference.

The activation of Akt kinase by a compound of the invention has utilityin the treatment of conditions resulting from reduced or insufficientAkt activity in a cell, including, but not limited to, ischemicinjuries. Suitable conditions resulting from reduced Akt activity fortreatment using the compounds and methods of the invention include, forexample, diseases or disorders characterized by insufficientvascularization (e.g., diabetic ulcers, gangrene, wounds requiringneovascularization to facilitate healing, Buerger's syndrome,hypertension, conditions characterized by a reduction inmicrovasculature), and certain neurological diseases or disorders (e.g.,Parkinson's disease, Alzheimer's disease, depression, anxiety,manic-depressive psychosis, post traumatic stress disorder, mildcognition impairment (MCI), amyotrophic lateral sclerosis (ALS),Huntington's disease, spinocerebellar degenerative disease, multiplesclerosis (MS), Pick's disease, schizophrenia, anxiety neurosis,obsessive-compulsive neurosis, head trauma, spinal cord injury,cerebrovascular disorder, cerebrovascular dementia, asymptomatic braininfarction, polyglutamine disease, prion disease, corticobasalganglionic degeneration, progressive supranuclear palsy, AIDSencephalopathy, muscular dystrophy, diabetic neuropathy).

Other conditions resulting from reduced Akt activity that may be treatedusing the compounds and methods of the invention include, but are notlimited to, diabetic retinopathy, diabetic nephropathy, liver cirrhosis,alcoholic hepatitis, senile diseases characterized by a decrease inself-regenerating ability, non-metabolic bone diseases, metabolic bonediseases, joint diseases, periodontal diseases, cytomegalovirusinfection, rheumatoid arthritis, Lyme disease, gout, sepsis syndrome,hyperthermia, ulcerative colitis, enterocolitis, osteoporosis,periodontal disease, glomerulonephritis, chronic non-infectiousinflammation of the lung, sarcoidosis, smoker's lung, granulomaformation, fibrosis of the liver, fibrosis of the lung, transplantrejection, graft vs. host disease, chronic myeloid leukemia, acutemyeloid leukemia, neoplastic disease, asthma bronchiale, type I insulindependent diabetes mellitus, arteriosclerosis, atherosclerosis,psoriasis, chronic B lymphocyte leukemia, common variableimmunodeficiency, disseminated intravascular coagulation, systemicsclerosis, encephalomyelitis, lung inflammation, hyper IgE syndrome,cancer metastasis, cancer growth, adoptive immune therapy, acquiredrespiratory distress syndrome, sepsis, reperfusion syndrome,postsurgical inflammation, organ transplantation, and alopecia.

AKT-mediated disorders resulting from reduced AKT activity are alsodisclosed in US 2004/0122077; US 2006/0241168; and US 2007/0219139, thecontents of which are incorporated herein by reference.

The pharmaceutical preparations disclosed herein are prepared inaccordance with standard procedures and are administered at dosages thatare selected to reduce, prevent, or eliminate, or to slow or halt theprogression of, the condition being treated (See, e.g., Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., andGoodman and Gilman's The Pharmaceutical Basis of Therapeutics,McGraw-Hill, New York, N.Y., the contents of which are incorporatedherein by reference, for a general description of the methods foradministering various agents for human therapy). The compositions of acompound represented by the disclosed compounds can be delivered usingcontrolled or sustained-release delivery systems (e.g., capsules,biodegradable matrices). Exemplary delayed-release delivery systems fordrug delivery that would be suitable for administration of thecompositions of the disclosed compounds are described in U.S. Pat. No.5,990,092 (issued to Walsh); U.S. Pat. No. 5,039,660 (issued toLeonard); U.S. Pat. No. 4,452,775 (issued to Kent); and U.S. Pat. No.3,854,480 (issued to Zaffaroni), the entire teachings of which areincorporated herein by reference.

For preparing pharmaceutical compositions from the compounds of thepresent invention, pharmaceutically acceptable carriers can either besolid or liquid. Solid form preparations include powders, tablets,pills, capsules, cachets, suppositories, and dispersible granules. Forexample, the compounds of the present invention may be in powder formfor reconstitution at the time of delivery. A solid carrier can be oneor more substances which may also act as diluents, flavoring agents,solubilizers, lubricants, suspending agents, binders, preservatives,tablet disintegrating agents, or an encapsulating material. In powders,the carrier is a finely divided solid which is in a mixture with thefinely divided active ingredient.

In tablets, the active ingredient is mixed with the carrier having thenecessary binding properties in suitable proportions and compacted inthe shape and size desired.

The powders and tablets preferably contain from about one to aboutseventy percent of the active ingredient. Suitable carriers aremagnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin,dextrin, starch, gelatin, tragacanth, methylcellulose, sodiumcaboxymethylcellulose, a low-melting wax, cocoa butter, and the like.Tablets, powders, cachets, lozenges, fast-melt strips, capsules andpills can be used as solid dosage forms containing the active ingredientsuitable for oral administration.

Liquid form preparations include solutions, suspensions, retentionenemas, and emulsions, for example, water or water propylene glycolsolutions. For parenteral injection, liquid preparations can beformulated in solution in aqueous polyethylene glycol solution.

Aqueous solutions suitable for oral administration can be prepared bydissolving the active ingredient in water and adding suitable colorants,flavors, stabilizing agents, and thickening agents as desired. Aqueoussuspensions for oral administration can be prepared by dispersing thefinely divided active ingredient in water with viscous material, such asnatural or synthetic gums, resins, methylcellulose, sodiumcarboxymethylcellulose, and other well-known suspending agents.

The pharmaceutical composition is preferably in unit dosage form. Insuch form, the composition is subdivided into unit doses containingappropriate quantities of the active ingredient. The unit dosage formcan be a packaged preparation, the package containing discretequantities of, for example, tablets, powders, and capsules in vials orampules. Also, the unit dosage form can be a tablet, cachet, capsule, orlozenge itself, or it can be the appropriate amount of any of these inpackaged form. The quantity of active ingredient in a unit dosepreparation may be varied or adjusted from about 0.1 mg to about 1000.0mg, preferably from about 0.1 mg to about 100 mg. The dosages, however,may be varied depending upon the requirements of the patient, theseverity of the condition being treated, and the compound beingemployed. Determination of the proper dosage for a particular situationis within the skill in the art. Also, the pharmaceutical composition maycontain, if desired, other compatible therapeutic agents.

In general, the methods for delivering the disclosed compounds andpharmaceutical compositions of the invention in vivo utilizeart-recognized protocols for delivering the agent with the onlysubstantial procedural modification being the substitution of thecompounds represented by any one of the disclosed compounds for thedrugs in the art-recognized protocols.

The compounds of the present invention may be administered by any route,preferably in the form of a pharmaceutical composition adapted to such aroute, and would be dependent on the condition being treated. Thecompounds and compositions may, for example, be administeredintravascularly, intramuscularly, subcutaneously, intraperitoneally,orally or topically. It will be obvious to those skilled in the art thatthe following dosage forms may comprise as the active ingredient, eithercompounds or a corresponding pharmaceutically acceptable salt of acompound of the present invention. A preferred method of administrationfor the compounds of the invention is intravenous administration.

In some embodiments, the composition may be administered parenterallyvia injection. Parenteral administration can include, for example,intraarticular, intramuscular, intravenous, intraventricular,intraarterial, intrathecal, subcutaneous, or intraperitonealadministration. Formulations for parenteral administration may be in theform of aqueous or non-aqueous isotonic sterile injection solutions orsuspensions. These solutions or suspensions may be prepared from sterilepowders or granules having one or more of the carriers mentioned for usein the formulations for oral administration. The compounds may bedissolved in polyethylene glycol, propylene glycol, ethanol, corn oil,benzyl alcohol, sodium chloride, and/or various buffers (e.g., sodiumbicarbonate, sodium hydroxide).

For oral administration, the pharmaceutical compositions may be in theform of, for example, a tablet, capsule, suspension or liquid. Thecomposition is preferably made in the form of a dosage unit containing atherapeutically effective amount of the active ingredient. Examples ofsuch dosage units are tablets and capsules. For therapeutic purposes,the tablets and capsules can contain, in addition to the activeingredient, conventional carriers such as binding agents, for example,acacia gum, gelatin, polyvinylpyrrolidone, sorbitol, or tragacanth;fillers, for example, calcium phosphate, glycine, lactose, maize-starch,sorbitol, or sucrose; lubricants, for example, magnesium stearate,polyethylene glycol, silica, or talc; disintegrants, for example potatostarch, flavoring or coloring agents, or acceptable wetting agents. Oralliquid preparations generally in the form of aqueous or oily solutions,suspensions, emulsions, syrups or elixirs may contain conventionaladditives such as suspending agents, emulsifying agents, non-aqueousagents, preservatives, coloring agents and flavoring agents. Examples ofadditives for liquid preparations include acacia, almond oil, ethylalcohol, fractionated coconut oil, gelatin, glucose syrup, glycerin,hydrogenated edible fats, lecithin, methyl cellulose, methyl or propylpara-hydroxybenzoate, propylene glycol, sorbitol, or sorbic acid.

For topical use the compounds of the present invention may also beprepared in suitable forms to be applied to the skin, or mucus membranesof the nose and throat, and may take the form of creams, ointments,liquid sprays or inhalants, lozenges, or throat paints. Such topicalformulations further can include chemical compounds such asdimethylsulfoxide (DMSO) to facilitate surface penetration of the activeingredient. Suitable carriers for topical administration includeoil-in-water or water-in-oil emulsions using mineral oils, petrolatumand the like, as well as gels such as hydrogel. Alternative topicalformulations include shampoo preparations, oral pastes and mouthwash.

For application to the eyes or ears, the compounds of the presentinvention may be presented in liquid or semi-liquid form formulated inhydrophobic or hydrophilic bases as ointments, creams, lotions, paintsor powders.

For rectal administration the compounds of the present invention may beadministered in the form of suppositories admixed with conventionalcarriers such as cocoa butter, wax or other glyceride. For preparingsuppositories, a low-melting wax, such as a mixture of fatty acidglycerides or cocoa butter, is first-melted and the active ingredient isdispersed homogeneously therein, as by stirring. The molten homogeneousmixture is then poured into convenient sized molds, allowed to cool, andthereby to solidify.

Delivery can also be by injection into the brain or body cavity of apatient or by use of a timed release or sustained release matrixdelivery systems, or by onsite delivery using micelles, gels andliposomes. Nebulizing devices, powder inhalers, and aerosolizedsolutions are representative of methods that may be used to administersuch preparations to the respiratory tract. Delivery can be in vitro, invivo, or ex vivo.

For example, suitable dosages for a compound of the invention can befrom about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg toabout 100 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about0.01 mg/kg to about 1 mg/kg body weight per treatment. Determining thedosage for a particular agent, patient and ischemia orischemia-reperfusion injury is well within the abilities of one of skillin the art. Preferably, the dosage does not cause or produces minimaladverse side effects.

A therapeutically effective amount of a compound of the invention can beadministered alone, or in combination with one or more other therapeuticagents. Suitable therapeutic agents that are useful for treatingischemic injuries, which can be administered in combination with acompound of the invention, include, but are not limited to, calciumchannel blockers, beta blockers, nitroglycerin, aspirin,anti-inflammatory agents, natriuretic factors, vasodilators,thrombolytic and antithrombolic agents.

Thus, a compound of the invention can be administered as part of acombination therapy (e.g., with one or more other therapeutic agents).The compound of the invention can be administered before, after orconcurrently with one or more other therapeutic agents. In someembodiments, a compound of the invention and other therapeutic agent canbe co-administered simultaneously (e.g., concurrently) as eitherseparate formulations or as a joint formulation. Alternatively, theagents can be administered sequentially, as separate compositions,within an appropriate time frame, as determined by the skilled clinician(e.g., a time sufficient to allow an overlap of the pharmaceuticaleffects of the therapies). A compound of the invention and one or moreother therapeutic agents can be administered in a single dose or inmultiple doses, in an order and on a schedule suitable to achieve adesired therapeutic effect (e.g., a reduction in and/or inhibition ofjoint inflammation). Suitable dosages and regimens of administration canbe determined by a clinician and are dependent on the agent(s) chosen,pharmaceutical formulation and route of administration, various patientfactors and other considerations.

EXAMPLES Example 1 Synthesis of Compounds

Materials and Methods:

Synthesis of RLip-EA-OH (Lip-EA)

RLipoic Acid (RLip-OH, 1.00 g) was dissolved in dioxane. The solutionwas protected from direct light by covering the reaction flask withfoil. DIEA (0.845 mL) and DSC (1.24 g) were added sequentially and thereaction was stirred vented overnight at room temperature to formLip-NHS in situ. An aqueous solution of glutamyl-alanine (H-EA-OH, 1.11g) and DIEA (2.65 mL) was prepared and added to the solution of Lip-NHS.The combined solution was stirred overnight and then transferred to areparatory funnel. Ethyl acetate followed by 5% KHSO4 (aq) was added tothe reaction mixture. The organic phase was collected and washed with 5%KHSO4 (aq) followed by saturated NaCl (aq). The organic phase was againcollected, dried with anhydrous Na2SO4 and after filtration evaporatedin vacuo to yield crude RLip-EA-OH as a light yellow foam. A portion ofthe crude foam was dissolved for purification by RP-HPLC with 2:1water-acetonitrile (0.5% HOAc) and the product isolated on a YMC PackPro C18 reverse phase column using a gradient of increasing acetonitrile(0.5% acetic acid) in water (0.5% acetic acid). Product containingfractions were identified by analytical HPLC, pooled, frozen, andlyophilized to provide RLip-EA-OH (165 mg) at 100% HPLC purity (area %at 220 nm). The product NMR was consistent with structure and had anobserved mass of 405 (M-1), calculated 406.

R/SLip-EA-OH, and SLip-EA-OH were prepared using a similar procedure.Optically pure RLip-OH and SLip-OH starting materials were obtained forthe preparation of RLip-EA-OH and SLip-EA-OH, respectively. Opticalpurity was assayed by optical rotation and met release specifications.Product structure and identity was confirmed by MS, HPLC retention timeshift (relation to Lip-OH starting material) and in most cases NMR.Compound structures, names, and appropriate abbreviations for thecompounds described herein are contained in Table 1. Analytical data onthe compounds described in this Example are shown in Table 2.

RLip-OH was obtained commercially (Labochim, Milan, Italy). R/SLip-Ea-OHand Ac-EA-OH were obtained by contract.

Preparation of the Dilysine Salt of RLip-EA-OH (Lip-EA)

RLip-EA-OH (20.0 g) was dissolved in ethanol water (19:1). Twoequivalents of lysine (14.4 g) were added to the ethanolic solution ofRLip-EA-OH and the slurry was warmed to reflux. After refluxing for 30minutes, the solution was allowed to cool to room temperature. Theproduct dilysine salt of RLip-EA-OH was recovered by filtration, rinsedwith absolute ethanol and dried to a constant weight for a 96% recoveryof dilysine salt of RLip-EA-OH.

TABLE 1 Compound Abbreviations, Structures, and Chemical Names. CompoundAbbreviations Compound Structure Full Name* RLip-OH

(R)-lipoic acid RLip-LGlu-LAla-OH; RLip-EA-OH

N-(R)-lipoyl-L-glutamyl-L-alanine R/SLip-LGlu-LAla-OH; R/SLip-EA-OH

N-(R/S)-lipoyl-L-glutamyl-L- alanine SLip-LGlu-LAla-OH; SLip-EA-OH

N-(S)-lipoyl-L-glutamyl-L-alanine R/SLip-LGlu-DAla-OH; R/SLip-Ea-OH

N-(R/S)-lipoyl-L-glutamyl-D- alanine Ac-LGlu-LAla-OH; Ac-EA-OH

N-acetyl-L-glutamyl-L-alanine *Standard nomenclature was used fornatural amino acids and common analogs [J. Biol. Chem. 1972,247:977-983]; Lipoyl = 1,2-dithiolane-3-pentanoyl

TABLE 2 Analytical Data for Compounds Listed in Table 1. HPLC MassOptical Compound NMR* Purity^(#) Spectroscopy Rotation RLip-OH^(†) NA99.9% NA +121.7 RLip-LGlu-LAla-OH Dithiolane —CH—S— m,  100% Calc: 406+36.1 1H, δ 3.10 Glutamyl, Found (M − 1): 405 Alaninyl αC—H m, 2H, δ4.38 R/SLip-LGlu-LAla-OH^(†) Dithiolane —CH—S— m, 99.6% Calc: 406 −24.21H, δ 3.10 Glutamyl, Found: 406 Alaninyl αC—H m, 2H, δ 4.38SLip-LGlu-LAla-OH NA 96.5% Calc: 406 −69.5 Found (M − 1): 405R/SLip-LGlu-DAla-OH^(†) NA 99.6% Calc: 406 NA Found: 406Ac-LGlu-LAla-OH^(†) NA 99.6% Calc: 261 NA Found: 261 *¹H NMR obtained ineither Methanol (d₄) or DMSO (d₆) ^(#)Area percent at λ = 220 nm^(†)Purchased commercially or via contract, analytical data fromcertificate of analysis. NA = not available

Example 2 RLip-EA-OH Treatment Induces Akt Phosphorylation/Activation ina Dose Dependent Manner in Cultured Cells

Aid is activated via phosphorylation by activation of signaltransduction pathways through known receptors in the plasma membrane ofcells. Phosphorylated Akt is readily detected with specific antibodiesin situ in fixed and permeabilized cells.

Materials and Methods:

Cytoblot Assay for Akt Activation

The ability of RLip-EA-OH to increase phosphorylated Akt was assayedusing an in-cell western blot, or cytoblot. A549 cells (human non-smallcell lung cancer cell line) were selected because these cells can bemanipulated to increase or decrease the level of phosphoryated Akt. Thecells were inoculated onto culture plates and allowed to adhere to thebottom, then treated, fixed, and permeabilized. Followingpermeabilization, the cells were treated with antibodies specific foreither phospho-Akt or total Akt. The cells were then treated withfluorescent secondary antibodies to quantify the amount of bound primaryantibody. Total Akt and phospho-Akt were simultaneously detected.

A549 cells were plated in 384 well black-wall, clear-bottom, cellculture-treated microtiter plates at 70% confluence. Cells wereincubated overnight to allow cell attachment. The media was changed tolow serum (0.1%, fetal bovine serum [FBS]) and the cells were incubatedfor another 24 hours. Cells were treated with test compounds, fixed andpermeabilized for the Akt assay. The fixative was 3.7% formaldehyde inphosphate buffered saline. The permeabilization buffer was 0.5% TritonX-100 in phosphate buffered saline. After permeabilization of cells, theamounts of total Akt and phosphorylated Akt at either of the twophosphorylation sites (threonine-308 and serine-473) were determined at7 test concentrations, each performed in quadruplicate. The phospho-Aktdata were normalized to total Akt and background subtracted foranalysis.

Following overnight serum starvation to reduce basal Aktphosphorylation, cells were stimulated with vehicle, RLip-EA-OH, orRLip-OH. Cells were treated for 45 minutes or 3 hours and studied forphosphorylation at serine-473 or threonine 308, respectively.

Immunohistochemistry Assay for Phosphorylated Akt

The ability of RLip-EA-OH to increase phosphorylated Akt was assayed inH9c2 cells by immunocytochemistry followed by visualization usingmicroscopy. H9c2 cells are a cell line derived from rat cardiac myocytesand can be manipulated to increase or decrease the level ofphosphoryated Akt. Cells were inoculated onto culture plates and allowedto adhere to the bottom, treated, fixed, and permeabilized. Followingpermeabilization, cells were treated initially with specific phospho-Aktantibodies followed by fluorescent secondary antibodies to quantify theamount of bound primary antibody.

H9c2 cells were plated in 12 well cell culture treated plates at 70%confluence. Cells were incubated overnight to allow cell attachment. Themedia was changed to low serum (0.5%, fetal bovine serum [FBS]) andcells incubated for another 48 hours. Cells were treated for 3 hourswith either vehicle, RLip-EA-OH (50 μM), or co-treatment with LY294002(25 μM), fixed with 3.7% formaldehyde in phosphate buffered saline andpermeabilized with 0.5% Triton X-100 in phosphate buffered saline. Afterpermeabilization, the cells were treated with antibody specific for Aktphosphorylated at threonine-308, followed by treatment with afluorescent labeled secondary antibody.

Result:

The effect of RLip-EA-OH and RLip-OH on Akt phosphorylation relative tototal Akt was assessed in A549 cells using a cytoblot assay asdescribed. A 3-fold and 2-fold increase in phosphorylated Akt at serine473 was observed following 45 minutes of treatment with RLip-EA-OH andRLip-OH, respectively (FIG. 2). Both RLip-EA-OH and RLip-OH increasedthe amount of phosphorylated Akt relative to total Akt in a dosedependent manner. RLip-EA-OH was more effective than RLip-OH at mostdose levels.

RLip-EA-OH treatment in the presence and absence of LY294002, a knownphosphotidylinositol-3′-kinase inhibitor (Vlahos, C. J., Matter, W. F.,Hui, K. Y., Brown, R. F. A specific inhibitor of phosphatidylinositol3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). JBiol Chem 1994, 269:5241-5248), was also evaluated. Cells were treatedfor 3 hours and studied for phosphorylation at tyrosine-308. IncreasedAkt phosporylation was observed following 3 hours of treatment withRLip-EA-OH alone. The increased phosphorylation of Akt in response toRLip-EA-OH treatment was completely inhibited by cotreatment with 5 μMLY294002 (FIG. 3).

In addition, the effect of RLip-EA-OH on Akt phosphorylation wasassessed in H9c2 cells using an immunohistochemistry assay as described.RLip-EA-OH treatment was compared to either vehicle treatment orRLip-EA-OH treatment in the presence of LY294002. Cells treated withvehicle showed little fluorescence. Fluorescence intensity was muchbrighter in cells that were treated for 3 hours with RLip-EA-OH.Co-treatment of cells with LY294002 for an additional 30 minutes priorto the addition of RLip-EA-OH diminished the fluorescence intensity fromAkt phosphorylation.

Example 3 RLip-EA-OH Activates Insulin Receptor Tyrosine Kinase (IRK)

Materials and Methods: IRK Activation Assay

Insulin receptor kinase activity was readily measured by a mobilityshift assay using a Caliper LabChip® 3000 and a 12-sipper LabChip® todetect both phosphorylated and unphosphorylated substrate (Caliper LifeSciences, Discovery Alliances and Services Division, Hanover, Md.). Themobility-shift kinase assay uses a microfluidic chip to measure theconversion of a fluorescent peptide substrate to a phosphorylatedproduct. The reaction mixture from a microtiter plate well wasintroduced through a capillary sipper onto the chip and thenonphosphorylated substrate and phosphorylated product were separated byelectrophoresis and detected via laser-induced fluorescence. Thesignature of the fluorescence signal over time revealed the extent ofthe reaction. The catalytic subunits of tyrosine kinases have endogenouslevels of activity in the presence of ATP and substrate, thus theresults are presented as a % of control.

Specifically, each test compound was diluted to 25 times its assayconcentration with 100% DMSO and added to 12 μL of an assay buffersolution (100 mM HEPES, 10 mM MnCl2, 5 mM B-GP, and 0.002% Brij)containing dithiotheritol (DTT, 2 mM) and insulin receptor kinase domain(Millipore catalog #14-466, 80 nM) prior to pre-incubation at roomtemperature for 15 minutes. Following the pre-incubation, assay buffer(12 μL) containing 304 of a fluorescent peptide substrate and ATP(162004) was added and the mixture further incubated at room temperaturefor an additional 1.5 hours at which point approximately 50% of thesubstrate was converted to product. The samples were placed on theLabChip 3000 to measure the amount of parent substrate andphosphorylated product.

Results:

Using the IRK activation assay described above, compounds were evaluatedat 100 μM for their ability to activate the insulin receptor tyrosinekinase (IRK). The results of this assay are shown in Table 3.

TABLE 3 The Effect of Compounds at 100 μM on the Activity of the InsulinReceptor Kinase. AVG % activity above Compound control (±SEM) RLip-EA-OH78 ± 15 R/SLip-EA-OH 14 ± 1  SLip-EA-OH  4 ± 12 R/SLip-Ea-OH 1 ± 6

Example 4 RLip-EA-OH Activates IGF1R Kinase and Src Tyrosine Kinase

Materials and Methods: IGF1 and Src Tyrosine Kinase Activation Assay.

IGF1R and Src tyrosine kinases have endogenous levels of activity in thepresence of ATP and substrate. This activity was readily measured usinga mobility shift assay using a Caliper LabChip® 3000 and a 12-sipperLabChip® to detect both phosphorylated and unphosphorylated substrate(Caliper Life Sciences, Discovery Alliances and Services Division).Enzyme, substrate and ATP concentrations were optimized for each assay.For the IGF1R assay, the final concentrations of the enzyme, peptide andATP were 20 nM, 1.5 μM, and 1220 μM, respectively. For the Src assay,the final concentrations of the enzyme, peptide and ATP were 2.5 nM, 1.5μM, and 17 μM, respectively.

RLip-EA-OH was evaluated at multiple concentrations for an effect onactivation of IGF1 receptor kinase (IGF1R) and Src. The effects of a 100μM concentration of different Lip-EA compounds on kinase activation areshown in Table 4a. The compounds tested activated IGF1R and Src withdifferent selectivities.

The effect of 300 μM RLip-EA-OH or RLip-OH on activation of differenttyrosine kinases was also assessed (Table 4b). At this concentration,RLip-EA-OH induced a significantly greater activation of both IRK andSrc tyrosine kinases than RLip-OH, relative to vehicle control.

TABLE 4a The Effect of Compounds at 100 μM on the Activity of IGF1R andSrc kinases. IGF1R Src % activity above control % activity above controlCompound (±SEM) (±SEM) RLip-EA-OH 42 ± 1 70 ± 1 R/SLip-EA-OH 27 ± 3 43 ±1 R/SLip-Ea-OH  17 ± 12 29 ± 1 SLip-EA-OH  −4 ± 10 19 ± 1

TABLE 4b The Effect of RLip-EA-OH and RLip-OH at 300 μM on the activityof IRK, IGF1R and Src Tyrosine Kinases. The data are the % increase inactivity above vehicle control. % Increase in activity at 300 μMTyrosine Kinase RLip-EA-OH R-α-Lip-OH IRK 77 ± 7 48 ± 1 IGF1R 33 ± 3 37± 4 Src 78 ± 2 15 ± 1

Example 5 RLip-EA-OH Prevents Apoptosis and Promotes Cell Survival inCultured Cells

Materials and Methods: Cell Survival Assay in Jurkat Cells

The ability of RLip-EA-OH to prevent apoptosis and promote cell survivalwas assessed using Jurkat cells deficient in Receptor-InteractingProtein (RIP), a cell death mediating protein. The Jurkat cell line isderived from human T-Lymphocytes. RIP-deficient Jurkat cells aresusceptible to apoptosis when treated with tumor necrosis factor alpha(TNFα). These cells were treated with either vehicle or RLip-EA-OH andthen apoptosis was triggered with TNFα. Cell survival was assessed toevaluate if RLip-EA-OH protected the cells against TNFα inducedapoptosis.

RIP-deficient Jurkat cells were seeded into 96 well plates at 20,000cells per well and treated for 2 hours with RLip-EA-OH (6 wells for eachconcentration) or DMSO. After pretreatment, 3 wells for each dose ofdrug were exposed to 10 ng/mL human recombinant TNFα (TNFα was not addedto the other 3 wells). Twenty four hours after TNFα treatment, ATP cellviability was determined (CellTiter-Glo, Promega) and the values wereused to calculate % survival of the cells.

In the absence of RLip-EA-OH, approximately 20% of the cells did notsurvive TNFα treatment. Treatment of cells with RLip-EA-OH preventedTNFα induced cell death in a dose dependent manner (Table 5).

TABLE 5 The effect of RLip-EA-OH on TNFα-induced apoptosis inRIP-deficient Jurkat cells. RLip-EA-OH (μM) 0* 0.006 0.012 0.05 0.1 0.21.5 % Survival (%) 82 84 95 96 98 101 106 SD 0.1 3 7 5 3 6 3 *Control

Example 6 RLip-EA-OH Inhibits Carbachol-Stimulated Increases inIntracellular Calcium in a Dose-Dependent Manner in Cultured Cells

Materials and Methods: Cytosolic Calcium Overload Assay

Cytosolic calcium increases in the CHO M1-WT3 cells when stimulated withcarbachol ([Molecular Devices, FlexStation Application Note 2,Comparison of FLIPR® and FLEXstation™ for Calcium Mobilization Assays])and may be detected with a fluorescent dye that binds calcium. Anincrease in fluorescence following carbachol stimulation is interpretedas an increase in cytosolic calcium. Chinese hamster ovary (CHO) cellswere allowed to adhere to the bottom of a 96-well culture plate. Thefluorescent dye and a test sample were placed on the plate and allowedto be taken up by the cells. The fluorescence level was measured everytwo seconds using a plate reader (Flexstation II, Molecular Devices,Sunnyvale Calif.). The cells were stimulated with carbachol andflourescent data reported as the peak increase in fluorescence abovebaseline following carbachol stimulation. Data were normalized to thepeak carbachol response in the control sample.

CHO cells from the cell line CHO-M1-WT3 were grown in Hams F12 mediumsupplemented with 10% FBS and 5 μg/mL G418 to maintain expression of theM1 muscarinic receptor. Cells were seeded the night before theexperiment at a concentration of 30,000 cells/well in a volume of 100 μLper well of black walled, clear bottomed, 96-well microplates (“assayplates”). Cells were incubated at 37° C. and 5% CO₂ overnight. The nextday, the cells were incubated at 37° C. for 60 minutes with Fluo-4 NW orCalcium-3 in Hank's Balanced Salt Solution along with 2.5 mM watersoluble probenecid and the test compound at the indicated concentrationsor vehicle. The final volume in each well was 200 μL. The cells wereplaced into the FlexStation system to monitor fluorescence before andafter the addition of 50 μL of 1 μM carbachol for a final concentrationof 200 nM. Fluorescence was measured for 17 seconds prior to carbacholaddition and 43 seconds following carbachol addition. The Fluo-4 dye wasexcited at a wavelength of 485 nm and emission measured at 525 nm. TheCalcium-3 dye was excited at a wavelength of 494 nm and emissionmeasured at 525 nm. The calcium response was reported as the peakfluorescence minus the baseline fluorescence calculated as the averageof the fluorescence prior to carbachol addition.

Result:

The ability of RLip-EA-OH, RLip-OH and Ac-EA-OH to prevent calciumoverload in CHO M1-WT3 cells was determined. The peak rise in cytosoliccalcium of CHO cells expressing muscarinic M1 receptor was measured inresponse to carbachol stimulation. RLip-EA-OH diminished the flux incytosolic calcium in a dose dependent manner, whereas RLip-OH andAc-EA-OH had minimal effect (FIG. 4). RLip-EA-OH has an inhibitoryeffect on carbachol stimulated increases in cytosolic calcium and theinhibition is dose dependent. RLip-OH and Ac-EA-OH had only modestcytosolic calcium diminishing activity.

Example 7 RLip-EA-OH Demonstrates a Greater Peroxyl Radical AbsorbanceCapacity than RLip-OH

Materials and Methods: Oxygen Radical Absorbance Capacity (ORAC) Assay

Peroxyl radicals are one species of reactive oxygen produced by cellsduring reperfusion. The presence of peroxyl radicals can be detected byfluorescein oxidation. In the presence of peroxyl radicals, fluoresceinfluorescence will decay over time. In the presence of an oxygen radicalscavenger, the rate of decay is diminished. The change in the rate ofdecay between control and in the presence of scavengers is used tomeasure the peroxyl radical scavenging ability of a test compound.

Each compound tested was diluted to a concentration ranging from 25 μMto 250 IJM in 10 mM phosphate buffer (pH=7.4) containing 10 nMfluorescein. The buffer and compound were incubated at 37° C. for 10minutes. After the incubation, flourescein fluorescence was measuredusing a plate reader (Molecular Devices Flexstation II, Ex=485, Em=520).Baseline fluorescence measurements were recorded for 15 minutes before2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH) was added.The fluorescence decay was recorded for 90 minutes. The fluorescencedecay without compound was subtracted from the fluorescence decay withcompound. The slope of the decay vs. concentration is reported as theabsorbance capacity.

Results:

The peroxyl radical absorbance capacity for lipoyl-containing compoundswas determined (Tables 6a and 6b). RLip-EA-OH displayed a greaterperoxyl radical absorbance capacity than RLip-OH. The results alsoindicate that the- lipoyl moiety is critical for scavenging peroxylradicals in the ORAC assay, as acetyl-glutamylalanine had no appreciableperoxyl radical absorption capability.

TABLE 6a ORAC assay results relative to RLip-OH. Compound ORAC ValueRLip-OH 100 ± 6 * RLip-EA-OH 131 ± 6  Ac-EA-OH  8 ± 5 † Values are themean of 3-4 experiments.

TABLE 6b ORAC assay results relative to RLip-EA-OH Compound ORAC ValueRLip-EA-OH 100 ± 11 R/SLip-EA-OH 102 ± 10 SLip-EA-OH 89 ± 4 Values arethe mean of 3-4 experiments.

Example 8 RLip-EA-OH Protects the Myocardium AgainstIschemia-Reperfusion Injury In Vivo

Materials and Methods: Rat Model of MI/R Injury

A rat model of MI/R injury was used as an in vivo screen to determine ifcertain lipoic acid derivative compounds are cardio-protective (e.g.,against mycoardial ischemia-reperfusion injury). This model is analogousto the ischemia-reperfusion injury observed in cardiac patientsfollowing coronary occlusions and cardiac surgery procedures, such ascoronary artery bypass grafting (CABG) (Matsui, T., Tao, J., del Monte,F., Lee, K.-H. et al., Akt Activation Preserves Cardiac Function andPrevents Injury After Transient Cardiac Ischemia In vivo, Circulation2001, 104:330).

General Procedure

The circumflex branch of the left coronary artery was ligatedtemporarily to induce regional ischemia in the left ventricular mass,followed by the injection of fluorescent microspheres to delineate theischemic region. The animals were sacrificed about 24 hours afterreperfusion and the hearts were excised, sectioned and stained withtriphenyltetrazolium. The direct impact of the pharmacologicintervention was determined by measuring the myocardial infarct area(MI), the ischemic area at risk (AR) and the left ventricular area (LV).The reduction of MI over the AR (MI/AR ratio) was used as the primarymeasure of drug efficacy relative to vehicle controls.

Detailed Procedure

Male Sprague-Dawley rats between 300 and 350 gm were used for theseexperiments. Anesthesia was induced with 3-4% isoflurane in an inductionchamber. After induction, anesthesia was maintained at a surgical planewith 1.5-2.0% isoflurane, administered by a Rodent Ventilator through a16-gauge angiocatheter introduced orally into the trachea. Theventilator was set at 2.5 cc at a rate of 60-65 breaths per minute tomaintain ventilation during surgery. The core temperature of the animalwas monitored and maintained at 37° C. using a rectal probe and aheating lamp attached to a temperature controller.

A left anterior thoracotomy was performed and the heart was exposedusing a vertical pericardotomy. The circumflex branch of the leftcoronary artery (LCx) was ligated approximately 4 mm from the aortausing a cardiovascular 7.0 monofilament suture on an 11 mm needle toinduce ischemia in the left ventricle.

Fluorescent microspheres (300 μL) were injected into the leftventricular cavity 10-20 minutes after the ligation to delineate theischemic area. The suture was removed 30 minutes after ligation toreperfuse the ischemic area and the ischemic area was checked forreperfusion.

The chest was then closed in layers using absorbable suture (Dexon 5-0)for the muscle layers and monofilament Nylon 5-0 suture was used toclose the cutaneous layer. The animals were allowed to recover, thenwere returned to the colony.

Twenty-four hours after reperfusion, anesthesia was induced usingketamine hydrochloride and the chest was opened. The animals weresacrificed with 15% potassium chloride aqueous solution (w/v) injectedinto the LV cavity to arrest the heart in diastole. The heart wasexcised distal to the aortic valve and washed with saline to remove theblood. Sagittal slices of the heart were obtained between the base ofthe ventricle and the apex. Five slices of heart tissue were obtained,each 2 mm thick. The slices were immersed in a 1%2,3,5-triphenyl-2H-tetrazolium chloride (TTC) in saline solution andthen stored in the dark for 30 minutes to stain.

Images of the slices were obtained under bright field (to observe theTTC staining) and under fluorescence (to observe the microspheres). Thearea at risk was determined by the absence of microspheres and theinfarct area was determined by the absence of TTC staining.

Result:

A meta analysis of RLip-EA-OH treated animals (n=64) vs. salinevehicle-treated (n=54) animals in the myocardial ischemia-reperfusionmodel demonstrated that RLip-EA-OH administered as an intraventricularcavity injection (1 mg/kg IC), effectively reduced the myocardialinfarct (MI) size relative to the area at risk (AR). The MI:AR ratiosfor vehicle and RLip-EA-OH treated animals were 0.373 (n=54) and 0.250(n=64), respectively corresponding to a 33% difference between groups(p<0.001)(FIG. 5). A significant reduction in the area of cardiac damagewas observed in myocardial tissue sections following RLip-EA-OHtreatment.

The timing of administration of RLip-EA-OH was investigated in the ratmyocardial ischemia-reperfusion model. RLip-EA-OH was administered at 1mg/kg IC pre-ischemia (15 minutes pre-occlusion), intra-ischemia (15minutes after occluding), or post-ischemia (within 1 minute afterreperfusion). RLip-EA-OH significantly (p<0.05) reduced myocardialtissue death pre-occlusion (38%), during occlusion (24%), and atreperfusion (32%) compared to those animals receiving saline vehicle(FIG. 6). RLip-EA-OH was effective at 1 mg/kg IC in reducing myocardialdamage whether administered prophylactically or therapeutically. Inaddition, RLip-EA-OH was effective at various dosages when administeredintravenously at 15 minutes pre-occlusion (FIG. 7). These resultsindicate that RLip-EA-OH administration effectively decreases the damageto the heart due to ischemia-reperfusion injury.

Example 9 The RLip-EA-OH Enantiomer is More Effective than the ParentMoiety RLip-OH, the Racemic Mix R/SLip-EA-OH, and SLip-EA-OH Enantiomerfor Reducing Myocardial Ischemia/Reperfusion (MI/R) Injury

The rat model of myocardial ischemia-reperfusion (MI/R) injury describedin Example 8 herein was employed to compare the efficacy of the pureRLip-EA-OH enantiomer with the efficacy of the parent moiety RLip-OH,pure SLip-EA-OH and the racemic mixture R/SLip-EA-OH in the treatment ofMI/R injury.

Results:

A meta analysis of animals treated with either RLip-OH (2 mg/kg IC) orRLip-EA-OH (1 mg/kg IC) is shown in Table 7 as a % reduction compared toa saline vehicle control. Ac-EA-OH, a non-lipoyl-containing compound,was also evaluated and found to be statistically similar to vehicle(data not shown). This result demonstrates that cardioprotection from IRinjury following treatment with RLip-EA-OH was better than treatmentwith RLip-OH.

TABLE 7 An Efficacy Comparison of RLip-OH to RLip-EA-OH Examined in RatModel of Myocardial Ischemia-Reperfusion Injury. Reduction of MI:ARRatio Compound Relative to Vehicle (%) RLip-EA-OH 31 ± 3* RLip-OH 19 ±7^(# ) *Results based upon a meta analysis of RLip-EA-OH treated animals(n = 75) vs. vehicle-treated (n = 89) animals ^(#)Results based upon ameta analysis of RLip-OH treated animals (n = 18) vs. vehicle-treated (n= 19) animals

An analysis of animals treated with either RLip-EA-OH (1 mg/kg IC) orR/SLip-EA-OH (1 mg/kg IC) is shown in Table 8 as a % reduction comparedto a saline vehicle control. This result demonstrates thatcardioprotection from IR injury following treatment with RLip-EA-OH wasbetter than treatment with R/SLip-EA-OH.

TABLE 8 An Efficacy Comparison of RLip-EA-OH to R/SLip-EA-OH Examined inRat Model of Myocardial Ischemia-Reperfusion Injury. Reduction of MI:ARRatio Compound Relative to Vehicle (%) RLip-EA-OH 31 ± 3* R/SLip-EA-OH19 ± 5^(# ) *Results based upon a meta analysis of RLip-EA-OH treatedanimals (n = 75) vs. vehicle-treated (n = 89) animals ^(#)Results basedupon a meta analysis of R/SLip-EA-OH treated animals (n = 26) vs.vehicle-treated (n = 25) animals

The results of study comparing the single isomer compounds RLip-EA-OHand SLip-EA-OH is shown in Table 9. Compounds were administered ateither 1 mg/kg or 2 mg/kg with a single bolus (IC) 15 minutes prior tothe ischemic episode. A racemic mixture was prepared by adding equalamounts of RLip-EA-OH and SLip-EA-OH. Racemic R/SLip-EA-OH was alsoevaluated. Data is presented as the reduction of myocardial infarct (MI)size relative to the area at risk (AR). This result demonstrates thatcardioprotection from IR injury following treatment with RLip-EA-OH wasbetter than treatment with the corresponding SLip-EA-OH.

TABLE 9 Reduction of myocardial infarct (MI) size relative to the areaat risk (AR) in Lip-EA-OH treated animals. Results based upon 9-10animals/group. Treatment Compound (mg/kg) MI/AR SEM RLip-EA-OH 2 0.2900.038 RLip-EA-OH + SLip-EA-OH 1 + 1 0.331 0.035 RLip-EA-OH 1 0.337 0.02SLip-EA-OH 2 0.400 0.042 SLip-EA-OH 1 0.401 0.026

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The teachings of all patents, published applications and referencescited herein are incorporated by reference in their entirety.

While this invention has been particularly shown and described withreferences to example embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1.-9. (canceled)
 10. A method of activating at least one cytoprotectivekinase in a cell, comprising contacting a cell with an effective amountof a compound represented by the following structural formula:

or a pharmaceutically acceptable salt thereof, wherein R¹ and R² in thecompound are each independently H or a hydrolyzable group, and whereinthe compound has a percent optical purity of at least 90% by weightrelative to the other stereoisomers.
 11. The method of claim 10, whereinthe hydrolyzable group is selected from the group consisting of(C₁-C₁₀)alkyl, (C₂-C₁₀)alkenyl, (C₂-C₁₀)alkynyl,(C₁-C₁₀)alkoxy(C₁-C₁₀)alkyl, (C₁-C₁₀)alkoxy(C₁-C₁₀)alkoxy(C₁-C₁₀)alkyl,aryl and aryl(C₁-C₁₀)alkyl, wherein each is optionally substituted with1 to 3 substituents selected from the group consisting of halo, nitro,cyano, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, amino,(C₁-C₆)alkylamino, di(C₁-C₆)alkylamino, (C₁-C₆)alkyl, halo(C₁-C₆)alkyl,(C₁-C₆)alkoxy, halo(C₁-C₆)alkoxy, morpholino, phenyl, and benzyl. 12.The method of claim 10, wherein the hydrolyzable group is selected fromthe group consisting of methyl, ethyl, propyl, butyl, pentyl, hexyl,heptyl, allyl, ethoxymethyl, methoxyethyl, methoxyethoxymethyl,methoxyethoxyethyl, benzyl, pentafluorophenyl, 2-N-(morpholino)ethyl,dimethylaminoethyl and para-methoxybenzyl.
 13. The method of claim 10,wherein the pharmaceutically acceptable salt comprises a monovalentcation or a divalent cation.
 14. The method of claim 13, wherein themonovalent cation is a monovalent metal cation and the divalent cationis a divalent metal cation.
 15. The method of claim 10, wherein thecompound is represented by the following structural formula:

or a pharmaceutically acceptable salt thereof.
 16. The method of claim10, wherein the compound is represented by the following structuralformula:


17. The method of claim 10, wherein the at least one cytoprotectivekinase is selected from the group consisting of Akt kinase, IRK kinase,IGF1R kinase, and Src kinase.
 18. The method of claim 10, wherein thecompound has a percent optical purity by weight of at least 99%.
 19. Themethod of claim 10, wherein the cell is in a subject.
 20. The method ofclaim 19, wherein the subject is a human.
 21. A method of inhibitingapoptosis in a subject in need thereof, comprising administering to thesubject an effective amount of a compound represented by the followingstructural formula:

or a pharmaceutically acceptable salt thereof, wherein R¹ and R² in thecompound are each independently H or a hydrolyzable group, and whereinthe compound or pharmaceutically acceptable salt thereof is at least 90%enantiomerically pure.
 22. A method of preventing cytosolic calciumoverload in a subject in need thereof, comprising administering to thesubject an effective amount of a compound represented by the followingstructural formula:

or a pharmaceutically acceptable salt thereof, wherein R¹ and R² in thecompound are each independently H or a hydrolyzable group, and whereinthe compound or pharmaceutically acceptable salt thereof is at least 90%enantiomerically pure.
 23. A method of increasing peroxyl radicalabsorbance in a subject in need thereof, comprising administering to thesubject an effective amount of a compound represented by the followingstructural formula:

or a pharmaceutically acceptable salt thereof, wherein R¹ and R² in thecompound are each independently H or a hydrolyzable group, and whereinthe compound or pharmaceutically acceptable salt thereof is at least 90%enantiomerically pure.